Diagnosis of Pulmonary Tuberculosis: Recent Advances and Diagnostic Algorithms

Diagnosis of Pulmonary Tuberculosis:

Recent Advances and Diagnostic Algorithms

Yon Ju Ryu, M.D.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Ewha Womans University School of Medicine, Seoul, Korea

Pulmonary tuberculosis (TB) persists as a great public health problem in Korea. Increases in the overall age of the population and the rise of drug-resistant TB have reinforced the need for rapid diagnostic improvements and new modalities to detect TB and drug-resistant TB, as well as to improve TB control. Standard guidelines and recent advances for diagnosing pulmonary TB are summarized in this article. An early and accurate diagnosis of pulmonary TB should be established using chest X-ray, sputum microscopy, culture in both liquid and solid media, and nucleic acid amplification.

Chest computed tomography, histopathological examination of biopsy samples, and new molecular diagnostic tests can be used for earlier and improved diagnoses, especially in patients with smear-negative pulmonary TB or clinically- diagnosed TB and drug-resistant TB.

Keywords: Lung; Tuberculosis; Diagnosis

control, both for treating the individual and for public health intervention to reduce further spread in the community

. Chest X-ray is useful but is not specific for diagnosing pul- monary TB. Moreover, TB can present with symptoms and atypical radiologic findings that are indistinguishable from those of community-acquired pneumonia

. As a result, it is not unusual for clinicians to prescribe a number of courses of antibiotics for pneumonia before the pulmonary TB is correctly diagnosed

. Therefore, an acid-fast bacilli (AFB) smear and bacteriological culture tests should be performed for patients with symptoms that are compatible with or sug- gestive of TB. However, mycobacterial culture, which has the highest sensitivity for diagnosing and confirming active TB, requires 2 to 6 weeks for interpretation

. Although sputum smear microscopy is a rapid, simple, and inexpensive tool for diagnosing pulmonary TB, it has low and variable sensitivity

. Recently, non-molecular and molecular assays have been developed for early detection of active TB with or without drug resistance detection. The diagnosis of TB is suspected from a combination of context, symptoms, clinical signs and investigations. Pulmonary TB refers to any bacteriologically- confirmed or clinically-diagnosed case of TB that involves the lung parenchyma or the tracheobronchial tree based on the revised previous standard case definitions for TB by the World Health Organization (WHO) in 2013

.

In this article, recent advances that allow better and earlier Copyright © 2015

The Korean Academy of Tuberculosis and Respiratory Diseases.

All rights reserved.

Introduction

Tuberculosis (TB) is a global health concern for both de- veloping and developed countries and has recently become more complex due to persistence in aging populations and the rise of drug-resistant strains, even in Korea

. In clinical practice, rapid TB diagnosis can be difficult, and early pulmo- nary TB detection continues to be challenging for clinicians.

Prompt diagnosis of active pulmonary TB is a priority for TB

Address for correspondence: Yon Ju Ryu, M.D.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Ewha Medical Center and Ewha Medical Research Institute, Ewha Womans University Mokdong Hospital, 1071 Anyangcheon-ro, Yangcheon-gu, Seoul 158-710, Korea

Phone: 82-2-2650-2840, Fax: 82-2-2650-2559 E-mail: [email protected]

Received: Jan. 29, 2015 Revised: Feb. 12, 2015 Accepted: Mar. 13, 2015

cc It is identical to the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/).

diagnosis of active pulmonary TB are summarized and diag- nostic algorithms in clinical practice are recommended, fo- cusing on the intermediate burden setting in Korea, based on updated literature reviews and standard guidelines

.

Diagnostic Methods

1. Radiologic study

Anyone with a cough that lasts for two weeks or more or with unexplained chronic fever and/or weight loss should be evaluated for TB

. Chest X-ray is the primary radiologic evalu- ation of suspected or proven pulmonary TB. Radiological presentation of TB may be variable but in many cases is quite characteristic. Radiology also provides essential information for management and follow-up of these patients and is ex- tremely valuable for monitoring complications. Chest X-ray is useful but is not specific for diagnosing pulmonary TB, and can be normal even when the disease is present

. Therefore, it cannot provide a conclusive independent diagnosis and needs to be followed by sputum testing. Many of the so-called unusual manifestations of adult TB are actually usual manifes- tations of primary disease. Post-primary TB in adults typically manifests as a heterogeneous, often cavitary opacity in the apical and posterior segments of the upper lobes and the su- perior segments of the lower lobes. Lymphadenopathy is rare.

Cavitation is the hallmark of post-primary TB and appears in about half of all patients. Patchy, poorly defined consolidation in the apical and posterior segments of the upper lobes and in the superior segment of the lower lobe is also commonly observed

. However, post-primary disease activity cannot be accurately assessed by chest radiography. Radiographic stability for 6 months and negative sputum cultures is the best indicator of inactive disease

. The descriptive terms ‘inactive’

or ‘old’ TB should be discarded in favor of radiographically sta- ble TB, as viable bacilli may persist despite adequate therapy.

Post-primary TB heals with parenchymal scarring and nod- ules. An important task for radiology is to determine whether these residual findings are indicative of active disease. For this, chest X-ray has limited value, since it can only establish that a lesion is stable, and stable lesions can contain active bacilli

.

Although chest X-ray is the primary diagnostic tool for evaluating pulmonary TB, chest computed tomography (CT) is generally required to detect fine lesions that can be overlooked on chest X-ray, to define equivocal lesions, or to evaluate complications

. Chest CT is an effective diagnostic method when plain films are normal or inconclusive, and it provides valuable information for managing the illness. Chest CT can add valuable information for detecting bacterial activ- ity. Branching opacities, cavitation, or consolidation are clear signs of active TB, but active disease must be confirmed by an- alyzing sputum for the presence of bacilli. A significant radio-

logic finding in chest CT is the “tree-in-bud” pattern, consisting of multiple branching linear structures that represent bron- chogenic dissemination of disease with caseating necrosis in the respiratory and terminal bronchioles

. These branching opacities have a lobar or segmental distribution and are con- sidered reliable markers of activity. Tree-in-bud opacities are also seen in other infections, but when visualized in combina- tion with cavitation or nodular opacities in the upper and pos- terior lung segments, and in the appropriate clinical setting, a specific diagnosis of pulmonary TB can be established

. While chest CT is useful for clarifying confusing findings, it has not been conclusively shown to have a significant impact on patient management

, therefore microbiological identifica- tion of TB by culture should follow this test

.

2. AFB smear microscopy and culture

For pulmonary TB, sputum is the most critical sample for laboratory testing. Direct sputum smear microscopy is the most widely used method for diagnosing pulmonary TB and is available in most primary health-care laboratories at the health-center level

. Smear microscopy may, however, be costly and inconvenient for patients, who must make multiple visits to health facilities and submit multiple sputum speci- mens over several days. Fortunately, good-quality microscopy of two consecutive sputum specimens has been shown to identify the vast majority (95%–98%) of smear-positive TB pa- tients

. The WHO policy on case detection by microscopy was, therefore, revised to recommend a reduction in the num- ber of specimens examined, from three to two in settings with appropriate external quality assurance and documented high- quality microscopy

. In addition, the WHO recommends that countries that have successfully implemented current WHO policies for a two-specimen case-finding strategy consider switching to same-day diagnosis, especially in settings where patients are likely to default from the diagnostic process

. The Korean guideline for TB recommends that presumptive TB patients should have multiple sputum samples, at least two, possibly three, and allows first sputum sample for front-loaded or same-day microscopy

.

Conventional light microscopy of Ziehl-Neelsen–stained

smears prepared directly from sputum specimens is the most

widely available test for diagnosing TB in resource-limited

settings. Ziehl-Neelsen microscopy is highly specific, but its

sensitivity is variable (20%–80%). Conventional fluorescence

microscopy is more sensitive (10%) than the Ziehl-Neelsen

and takes less time, but it is limited by the high cost of mercury

vapor light sources, the need for regular maintenance, and the

dark room requirement

. Light-emitting diodes (LED) have

been developed to offer fluorescence microscopy without

the associated costs. The efficacy of LED microscopy was as-

sessed by the WHO and evaluated by standards appropriate

for the accuracy and the effect of new TB diagnostics on both

patients and public health

. The results showed that the accu- racy of LED microscopy was equivalent to that of international reference standards; it was more sensitive than conventional Ziehl-Neelsen microscopy, and it had qualitative, operational, and cost advantages over both conventional fluorescence and Ziehl-Neelsen microscopy. Based on these findings, the WHO recommends that conventional fluorescence microscopy be replaced by LED microscopy and that LED microscopy be phased in as an alternative for conventional Ziehl-Neelsen light microscopy

.

Laboratory diagnosis of TB relies on direct microscopic examination of sputum specimens. However, the technique, although specific, has low and variable sensitivity and cannot identify drug-resistant strains. Clinicians have been advised to obtain culture confirmation of TB whenever possible. This not only confirms the diagnosis, but also obtains material for cru- cial drug susceptibility testing (DST)

. Mycobacterial cul- ture is more sensitive, but growth of TB bacilli on traditional solid medium requires 4–8 weeks, which delays appropriate treatment in the absence of a confirmed diagnosis. Culturing mycobacteria is mainly done on solid media, the Lowenstein- Jensen slope, or in broth media. These methods are slow, with cultures from microscopy-positive material taking from 2–4 weeks and for microscopy-negative material from 4–8 weeks.

Therefore, liquid media remains the mycobacteriology gold standard for initial isolation, because it is significantly faster (between 10 and 14 days) and is better for isolation, com- pared to solid media. For DST, the delay may be reduced to as little as 10 days compared to 4–6 weeks with conventional solid media. Liquid systems are more sensitive for detecting mycobacteria and may increase the case yield by 10% over solid media. With increased sensitivity and reduced delays, liquid systems may contribute significantly to improved pa- tient management. Liquid systems are, however, more prone to contamination by other microorganisms. In experienced laboratories, approximately 5%–10% of specimens fail to yield results because of contamination. Procedures to prevent cross-contamination (due to carryover of bacilli from posi- tive to negative specimens) should also be strictly followed, especially where increased numbers of positive specimens are processed in high-incidence countries

. Solid media are made of agar, egg, and malachite green to limit the growth of remaining contaminants (Lowenstein, Stonebrink, or Ogawa medium). Liquid culture and DST systems are more complex and sensitive than solid culture and DST media. Increased bacterial contamination and an increased frequency of nontu- berculous mycobacterial (NTM) isolation must be addressed.

A rapid method to differentiate the Mycobacterium tubercu- losis complex from other mycobacterial species is essential.

Several manufacturers have recently marketed tools that can automatically detect M. tuberculosis growth in the laboratory, such as the Bactec “Mycobacterial Growth Indicator Tube 960” (MGIT 960; Becton-Dickinson, Sparks, MD, USA) and the

MB/Bact Alert 10 3D (Biomérieux, Durham, NC, USA). Unfor- tunately, these automatized incubators are expensive, they do not give rapid mycobacterial species’ identification, and they do not identify contaminated or mixed cultures. Conversely, cultures on solid media provide all of this information with a simple observation of colonies. The current guideline rec- ommends that all specimens cultured on liquid media also be inoculated on solid media to ensure purity and sufficient strengthen for the diagnosis

.

3. Molecular methods

1) Nucleic acid amplification testing: Nucleic acid am- plification (NAA) tests are a reliable way to increase the speci- ficity of diagnosis, but the sensitivity is too poor to rule out disease, especially in smear-negative (paucibacillary) disease where clinical diagnosis is equivocal and where the clinical need is greatest

. Compared with AFB smear microscopy, the added value of NAA testing lies in (1) its greater posi- tive predictive value (PPV) (>95%) with AFB smear-positive specimens in settings in which NTMs are common and (2) its ability to rapidly confirm the presence of M. tuberculosis in 50%–80% of AFB smear-negative, culture-positive specimens.

Compared with culture, NAA tests can detect the presence of M. tuberculosis bacteria in a specimen weeks before culture for 80%–90% of patients suspected to have pulmonary TB whose TB is ultimately confirmed by culture

. Although NAA testing is recommended to perform the initial diagnosis of persons suspected to have TB, the currently available NAA tests should not be ordered routinely when the clinical suspi- cion of TB is low because the PPV of the NAA test is <50% for such cases

.

The United States’ Centers for Disease Control and Pre-

vention (CDC) recommends that NAA testing should be

performed on at least one respiratory specimen using a Food

and Drug Administration (FDA)–approved test

from each

patient that has signs and symptoms of pulmonary TB for

whom a diagnosis of TB is being considered but has not yet

been established and for whom the test result would alter case

management or TB control activities such as contact investi-

gations. A single negative NAA test result should not be used

as a definitive result to exclude TB, especially when clinical

suspicion of TB is moderate to high. Rather, a negative NAA

test result should be used as additional information in making

clinical decisions, to expedite testing for an alternative diagno-

sis, or to prevent unnecessary TB treatment. Clinicians should

interpret all laboratory results on the basis of the clinical situa-

tion

. The Korean guidelines for TB with intermediate setting

recommend that when an individual is suspected of having

pulmonary TB, NAA tests should be performed at least once

in combination with AFB smear microscopy and culture

.

NAA tests cannot replace culture and microscopy but should

be interpreted along with conventional tests and clinical data

for diagnosing TB. NAA tests are also not useful for monitoring treatment progress since they can detect non-viable bacteria and give false-positive results; however, they can distinguish M. tuberculosis from NTM

. Smear microscopy or clinical presentation cannot differentiate between TB and NTM, and the recovery rate of NTM from AFB smear-positive sputum specimens is steadily increasing in Korea. Therefore, NAA tests could be a useful diagnostic test for patients with AFB smear-positive sputum for rapidly detecting pulmonary TB and differentiating from NTM

.

2) Line probe assay: Conventional methods for mycobac- teriological culture, identification of an M. tuberculosis com- plex and DST are slow and cumbersome, therefore, rapid DST of isoniazid and rifampicin or of rifampicin alone using mo- lecular technologies is recommended over conventional test- ing in sputum smear-positive or culture proven cases at risk of multi-drug resistant (MDR)-TB, such as previously-treated patients

. Line probe assay (LPA) has generally been avail- able for this purpose in rapid DST and is a type of molecular assay that can allow specific gene markers associated with rifampicin resistance alone or in combination with isoniazid to be detected. Resistance to isoniazid occurs primarily due to mutations in katG, followed by mutations at the InhA active site (20%–35%) and in the promoter region of ahpC. Muta- tions in the rpoB region are found in about 96% of rifampicin -resistant M. tuberculosis isolates

.

LPA technology is performed in the following steps, includ- ing extracting DNA from mycobacterial isolates or directly from clinical specimens, polymerase chain reaction (PCR) amplification of the resistance-determining region of the gene, hybridization of labeled PCR products with oligonucleotide probes immobilized on a strip, and colorimetric development that allows for lines to be seen where the probes are located.

According to systematic reviews and meta-analyses to evalu- ate assay performance, results that compared conventional DST methods showed that LPA are highly sensitive (≥97%) and specific (≥99%) for detecting rifampicin resistance, alone or in combination with isoniazid (sensitivity ≥90%; specificity

≥99%), in M. tuberculosis isolates and in smear-positive spu- tum specimens

. However, LPA cannot replace conventional culture with DST, and mycobacteriological culture for smear- negative specimens with DST because second-line anti-TB drugs are still required.

3) Xpert MTB/RIF (Cepheid): The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA, USA; hereafter referred to as Xpert MTB/RIF) is a novel, rapid, automated, and cartridge-based NAA test that can detect TB along with rifampicin resistance directly from sputum within 2 hours of collection

. The Gen- eXpert cartridges are pre-loaded with all of the necessary re- agents for sample processing, DNA extraction, amplification, and laser detection of the amplified rpoB gene target. A major

advantage of the Xpert MTB/RIF test is that it can be accu- rately administrated with minimal hands-on technical time.

The sensitivity and specificity of this test has been reported to be acceptable for TB detection

. The Xpert MTB/RIF test is a valuable, highly sensitive, and specific new tool for early TB detection and for determining rifampicin resistance. While it should be noted that mono-resistance to rifampicin is found in approximately 5% of rifampicin-resistant strains, a high pro- portion of rifampicin resistance is associated with concurrent resistance to isoniazid (~95%). Thus, detecting resistance to rifampicin can be used as a marker for MDR-TB with a high level of accuracy

.

In a recent study of the performance of Xpert MTB/RIF, among the 561 culture-positive patients (561/1730), a single, direct Xpert MTB/RIF test identified 98.2% (551 out of 561) of the sputum smear-positive TB cases and 72.5% (124 out of 171) of those with sputum smear-negative TB. The test was specific in 604 of 609 patients (99.2%) not affected by TB. A second Xpert MTB/RIF test among patients with sputum smear-negative, culture-positive TB increased detection sen- sitivity by 12.6% and a third by 5.1%, to reach 90.2%. When compared to phenotypic DST, the Xpert MTB/RIF assay cor- rectly identified 97.6% (200 out of 205) of patients harboring rifampicin-resistant strains and 98.1% (504 out of 514) of those with rifampicin-susceptible strains

. The WHO issued initial recommendations on Xpert MTB/RIF, especially for in- dividuals suspected of having MDR-TB

. Xpert MTB/RIF has higher sensitivity for TB detection in smear-positive patients than in smear-negative patients; nonetheless, this test may be valuable as an add-on test following smear microscopy in patients previously found to be smear-negative

. Recently, the International Standards for TB Care recommended that Xpert MTB/RIF and/or sputum cultures should be performed in patients suspected of having pulmonary TB but that have negative sputum smears

. However, in settings or patient groups where rifampicin resistance is rare, the PPV of Xpert MTB/RIF is adversely affected, and is significantly lower when rifampicin resistance prevalence falls below 5%. Therefore, testing new TB cases that are not at risk of MDR-TB in low MDR-TB prevalence settings, including Korea, results in a low PPV, which requires rifampicin resistance confirmation by phenotypic DST or LPA (and not by a second Xpert MTB/

RIF test) prior to initiating treatment

. However, a recently

updated WHO policy recommends that a repeated Xpert

MTB/RIF test on a fresh specimen can be useful when Xpert

MTB/RIF detects M. tuberculosis with rifampicin resistance

in patients considered to be at low risk of MDR-TB

. The tests

are expensive, so careful consideration should be given in do-

mestic circumstances to the resource implications of offering

routine Xpert MTB/RIF testing. The current Korean guidelines

recommend that the Xpert MTB/RIF test be used in patients

suspected of having TB and are at high risk of MDR-TB, such

as previously-treated cases or human immunodefieciency

virus–positive TB and severe cases of pulmonary TB

.

Diagnostic Algorithms of Pulmonary Tuberculosis

1. Smear-positive pulmonary TB

People suspected of having TB should be referred for medi- cal evaluation. A posterior-anterior chest X-ray should be taken, and a chest X-ray that appears suggestive of TB should be followed by further diagnostic investigation. Multiple spu- tum samples (at least two, possibly three samples) that allow at least, first one front-loaded sputum specimen should, if pos- sible, be sent for TB microscopy and cultured for suspected respiratory TB before starting treatment

. Spontaneously produced sputum should be obtained if possible; otherwise, induction of sputum or bronchoscopy and lavage can be con- sidered

.

NAA tests can rapidly confirm TB diagnosis and distinguish M. tuberculosis from NTM in a sputum smear-positive per-

son. Positive results for both the NAA and the AFB smear tests strongly suggest that the patient has TB, and anti-TB treatment should be initiated while awaiting culture results. The PPV of FDA-approved NAA tests for TB in the United States is >95%

in AFB smear-positive cases. If the NAA result is negative and the AFB smear result is positive, a patient can be presumed to have an NTM infection

. Culture remains the gold standard for laboratory confirmation of TB and is required for isolating bacteria for DST and genotyping

.

2. Smear-negative pulmonary TB

Current TB diagnostics are still suboptimal in performance, especially for smear-negative TB. Because TB can present with many different symptoms, the first obstacle in diagnosing smear-negative TB is discerning the varied clinical presenta- tions, to determine which conditions are highly suspicious and should be included in the differential diagnosis. A di- agnostic approach to an AFB smear-negative patient with possible TB includes, where available, a detailed medical history and clinical examination as well as radiological, mi- crobiological, molecular, and histological investigations

. In patients suspected of having TB with smear-negative, cultur- ing remains essential for diagnostic testing and should be in- cluded when evaluating patients suspected of having TB with negative sputum smears. When performed correctly, culture increases diagnostic sensitivity, which ideally results in earlier case detection. The state-of-the-art diagnostic path often uses the tests mentioned above in combination, and additional diagnostic tools/procedures such as induced sputum

, chest CT imaging

, bronchoscopy with lavage

, and lung tis- sue biopsy

are sometimes performed.

NAA testing is recommended to perform the initial diagno- sis of patients that are suspected to have TB, even in smear- negative patients. If the NAA result is positive and the AFB smear result is negative, the clinician must consider the case as TB positive and begin anti-TB treatment while awaiting culture results

. If sputum smears and NAA tests are nega- tive, and TB is still suspected, cultures are the most sensitive tests for TB. Culture is therefore very useful for diagnosing both smear-negative TB and drug-resistant TB. Although new diagnostic methods can decrease turn-around time with con- siderable diagnostic accuracy and convenience, they are not a replacement for culture and conventional DST

. However, in certain TB cases that are caused by pauci-bacillary disease, where only a small number of M. tuberculosis organisms are present, cultures can be negative; in addition sampling error, or technical problems can occur. In cases where the culture is negative, the standard used to compare with the diagnostic test could be response to treatment, clinical features, or a posi- tive culture in the future. A TB diagnosis in this population would likely be achieved on a case-by-case basis and as such, would probably not be reported in many studies

.

Diagnosing TB worldwide still consists of methods that are intended to isolate the pathogen, which is a major limitation when the mycobacterial load is low or the site of infection is not easily accessible. For these reasons, diagnosis of smear- negative TB is often delayed, and such a diagnosis is often made based on the clinical response to empiric anti-TB treat- ment without microbiological confirmation

. Diagnostic algorithms have been recommended in the absence of rapid, simple, and accurate diagnostic tools for smear-negative pul- monary TB

. Diagnosing smear-negative pulmonary TB is achieved with at least two adequate expectorated multiple sputum samples, with negative smears, chest radiography find- ings consistent with TB, and a lack of response to a number of days of broad-spectrum antibiotics while awaiting culture results. For such patients, sputum cultures should be obtained and active follow-up is needed

. There are several limita- tions to using diagnostic steps based on antibiotics response.

Because the fluoroquinolones and aminoglycosides are active

against the M. tuberculosis complex and may cause transient

improvement in people with TB, they should be avoided in

this diagnostic phase. Strict adherence to the sequential steps

of the algorithm could delay appropriate treatment in patients

with an illness that is worsening rapidly. Finally, studies have

shown that patients with TB might respond, at least tran-

siently, to empirical broad-spectrum antibiotics, a frequent el-

ement of diagnostic algorithms

. Recently, the WHO revised

the TB definition to include clinically-diagnosed TB instead

of smear-negative TB. A clinically-diagnosed TB case is one

that does not fulfill the criteria for bacteriological confirmation

but has been diagnosed with active TB by a clinician or other

medical practitioner who has decided to give the patient a

full course of TB treatment. Clinically-diagnosed cases subse-

quently found to be bacteriologically-positive (before or after starting treatment) should be reclassified as bacteriologically confirmed

.

Clinicians who manage patients with suspected TB should ensure that their diagnostic practices align with the guidelines for TB, and use sputum smear microscopy and culture to investigate adult patients with suspected active TB

. Novel methods allow for rapid diagnosis of active TB in patients with negative sputum smears for AFB and enable prompt, highly accurate identification of drug-resistant strains of M.

tuberculosis directly from respiratory specimens. Some of the structural limits of current TB diagnostics are likely to be over- come by such new tools, but research is still needed. The suc- cess of implementing new technologies and the development of additional diagnostic approaches must take into account the diagnostic needs of each context (point-of-need testing approach) together with the logistic, economic and technical constraints that are present in the majority of intermediate- TB-burdened settings in Korea.

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

References

1. World Health Organization. Global tuberculosis report 2014 [Internet]. Geneva: World Health Organization; 2014 [cited 2015 Mar 1]. Available from: http://apps.who.int/iris/bitstre am/10665/137094/1/9789241564809_eng.pdf?ua=1.

2. Park YK, Park YS, Na KI, Cho EH, Shin SS, Kim HJ. Increased tuberculosis burden due to demographic transition in Korea from 2001 to 2010. Tuberc Respir Dis 2013;74:104-10.

3. World Health Organization. Early detection of tuberculo- sis: an overview of approaches, guidelines and tools [In- ternet]. Geneva: World Health Organization; 2011 [cited 2015 Mar 1]. Available from: http://www.who.int/iris/

handle/10665/70824#sthash.TN5snAtB.dpuf.

4. Woodring JH, Vandiviere HM, Fried AM, Dillon ML, Williams TD, Melvin IG. Update: the radiographic features of pulmo- nary tuberculosis. AJR Am J Roentgenol 1986;146:497-506.

5. Krysl J, Korzeniewska-Kosela M, Muller NL, FitzGerald JM.

Radiologic features of pulmonary tuberculosis: an assess- ment of 188 cases. Can Assoc Radiol J 1994;45:101-7.

6. Grossman RF, Hsueh PR, Gillespie SH, Blasi F. Community- acquired pneumonia and tuberculosis: differential diagnosis and the use of fluoroquinolones. Int J Infect Dis 2014;18:14- 21.

7. Wang M, Fitzgerald JM, Richardson K, Marra CA, Cook VJ, Hajek J, et al. Is the delay in diagnosis of pulmonary tubercu-

losis related to exposure to fluoroquinolones or any antibi- otic? Int J Tuberc Lung Dis 2011;15:1062-8.

8. World Health Organization. Definitions and reporting frame- work for tuberculosis: 2013 revision (updated December 2014) [Internet]. Geneva: World Health Organization; 2013 [cited 2015 Mar 1]. Available from: http://apps.who.int/iris/

bitstream/10665/79199/1/9789241505345_eng.pdf?ua=1.

9. National Institute for Health and Clinical Excellence. Tuber- culosis: clinical diagnosis and management of tuberculosis, and measures for its prevention and control. London: Na- tional Institute for Health and Clinical Excellence; 2011.

10. Wilson ML. Recent advances in the laboratory detection of Mycobacterium tuberculosis complex and drug resistance.

Clin Infect Dis 2011;52:1350-5.

11. Joint Committee for the Revision of Korean Guidelines for Tuberculosis, Korea Centers for Disease Control and Preven- tion. Korean guidelines for tuberculosis. 2nd ed. Seoul and Cheongwon: Joint Committee for the Revision of Korean Guidelines for Tuberculosis, Korea Centers for Disease Con- trol and Prevention; 2014.

12. International standards for tuberculosis care, 3rd ed [Inter- net]. The Hague: TB Care 1; 2014. Available from: http://www.

who.int/tb/publications/ISTC_3rdEd.pdf?ua=1.

13. Steingart KR, Schiller I, Horne DJ, Pai M, Boehme CC, Dendu- kuri N. Xpert(R) MTB/RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. Cochrane Database Syst Rev 2014;1:CD009593.

14. Lee KS, Song KS, Lim TH, Kim PN, Kim IY, Lee BH. Adult- onset pulmonary tuberculosis: findings on chest radiographs and CT scans. AJR Am J Roentgenol 1993;160:753-8.

15. Geng E, Kreiswirth B, Burzynski J, Schluger NW. Clinical and radiographic correlates of primary and reactivation tubercu- losis: a molecular epidemiology study. JAMA 2005;293:2740- 5.

16. Jones BE, Ryu R, Yang Z, Cave MD, Pogoda JM, Otaya M, et al. Chest radiographic findings in patients with tuberculosis with recent or remote infection. Am J Respir Crit Care Med 1997;156(4 Pt 1):1270-3.

17. Im JG, Itoh H, Shim YS, Lee JH, Ahn J, Han MC, et al. Pul- monary tuberculosis: CT findings: early active disease and sequential change with antituberculous therapy. Radiology 1993;186:653-60.

18. World Health Organization. Same-day diagnosis of tuberculo- sis by microscopy: WHO policy statement [Internet]. Geneva:

World Health Organization; 2011 [cited 2015 Mar 1]. Available from: http://www.who.int/iris/handle/10665/44603#sthash.

gvkTkPOD.dpuf.

19. Davis JL, Cattamanchi A, Cuevas LE, Hopewell PC, Steingart KR. Diagnostic accuracy of same-day microscopy versus standard microscopy for pulmonary tuberculosis: a system- atic review and meta-analysis. Lancet Infect Dis 2013;13:147- 54.

20. Steingart KR, Henry M, Ng V, Hopewell PC, Ramsay A, Cun-

ningham J, et al. Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review. Lan- cet Infect Dis 2006;6:570-81.

21. World Health Organization. Fluorescent light emitting diode (LED) microscopy for diagnosis of tuberculosis: policy state- ment [Internet]. Geneva: World Health Organization; 2010 [cited 2015 Mar 1]. Available from: http://www.who.int/tb/

laboratory/who_policy_led_microscopy_july10.pdf.

22. Kanchana MV, Cheke D, Natyshak I, Connor B, Warner A, Martin T. Evaluation of the BACTEC MGIT 960 system for the recovery of mycobacteria. Diagn Microbiol Infect Dis 2000;37:31-6.

23. Greco S, Girardi E, Navarra A, Saltini C. Current evidence on diagnostic accuracy of commercially based nucleic acid am- plification tests for the diagnosis of pulmonary tuberculosis.

Thorax 2006;61:783-90.

24. Ling DI, Flores LL, Riley LW, Pai M. Commercial nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis and meta-regression.

PLoS One 2008;3:e1536.

25. Centers for Disease Control and Prevention (CDC). Updated guidelines for the use of nucleic acid amplification tests in the diagnosis of tuberculosis. MMWR Morb Mortal Wkly Rep 2009;58:7-10.

26. Diagnostic Standards and Classification of Tuberculosis in Adults and Children. This official statement of the American Thoracic Society and the Centers for Disease Control and Prevention was adopted by the ATS Board of Directors, July 1999. This statement was endorsed by the Council of the In- fectious Disease Society of America, September 1999. Am J Respir Crit Care Med 2000;161(4 Pt 1):1376-95.

27. Kwon YS, Koh WJ. Diagnosis of pulmonary tuberculosis and nontuberculous mycobacterial lung disease in Korea. Tuberc Respir Dis 2014;77:1-5.

28. World Health Organization. Molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tu- berculosis (MDR-TB): policy statement [Internet]. Geneva:

World Health Organization; 2008 [cited 2015 Mar 1]. Avail- able from: http://www.who.int/tb/laboratory/lpa_policy.pdf.

29. Zhang Y, Yew WW. Mechanisms of drug resistance in Myco- bacterium tuberculosis. Int J Tuberc Lung Dis 2009;13:1320- 30.

30. Ramaswamy S, Musser JM. Molecular genetic basis of antimi- crobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuber Lung Dis 1998;79:3-29.

31. World Health Organization. Automated real-time nucleic acid amplification technology for rapid and simultaneous detec- tion of tuberculosis and rifampicin resistance: Xpert MTB/RIF assay for the diagnosis of pulmonary and extrapulmonary TB in adults and children: policy update [Internet]. Geneva:

World Health Organization; 2013 [cited 2015 Mar 1]. Available from: http://www.who.int/iris/handle/10665/112472#sthash.

WDSfafG9.dpuf.

32. Weyer K, Mirzayev F, Migliori GB, Van Gemert W, D’Ambrosio L, Zignol M, et al. Rapid molecular TB diagnosis: evidence, policy making and global implementation of Xpert MTB/RIF.

Eur Respir J 2013;42:252-71.

33. World Health Organization. Policy statement: automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF system [Internet]. Geneva: World Health Organization; 2011 [cited 2015 Mar 1]. Available from:

http://www.who.int/iris/handle/10665/44586#sthash.Cdd- BEYMy.dpuf.

34. Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010;363:1005-15.

35. World Health Organization. Xpert MTB/RIF implementation manual: technical and perational ‘how-to’: practical consid- erations [Internet]. Geneva: World Health Organization; 2014 [cited 2015 Mar 1]. Available from: http://apps.who.int/iris/

bitstream/10665/112469/1/9789241506700_eng.pdf.

36. Chan HS, Sun AJ, Hoheisel GB. Bronchoscopic aspiration and bronchoalveolar lavage in the diagnosis of sputum smear- negative pulmonary tuberculosis. Lung 1990;168:215-20.

37. Seong GM, Lee J, Lee JH, Kim JH, Kim M. Usefulness of spu- tum induction with hypertonic saline in a real clinical prac- tice for bacteriological yields of active pulmonary tuberculo- sis. Tuberc Respir Dis 2014;76:163-8.

38. Colebunders R, Bastian I. A review of the diagnosis and treat- ment of smear-negative pulmonary tuberculosis. Int J Tuberc Lung Dis 2000;4:97-107.

39. Siddiqi K, Lambert ML, Walley J. Clinical diagnosis of smear- negative pulmonary tuberculosis in low-income countries:

the current evidence. Lancet Infect Dis 2003;3:288-96.

40. Lee JJ, Chong PY, Lin CB, Hsu AH, Lee CC. High resolution chest CT in patients with pulmonary tuberculosis: character- istic findings before and after antituberculous therapy. Eur J Radiol 2008;67:100-4.

41. Chawla R, Pant K, Jaggi OP, Chandrashekhar S, Thukral SS.

Fibreoptic bronchoscopy in smear-negative pulmonary tu- berculosis. Eur Respir J 1988;1:804-6.

42. Lee HS, Park KU, Park JO, Chang HE, Song J, Choe G. Rapid, sensitive, and specific detection of Mycobacterium tuberculo- sis complex by real-time PCR on paraffin-embedded human tissues. J Mol Diagn 2011;13:390-4.

43. Park JS, Kang YA, Kwon SY, Yoon HI, Chung JH, Lee CT, et al.

Nested PCR in lung tissue for diagnosis of pulmonary tuber- culosis. Eur Respir J 2010;35:851-7.

44. International standards for tuberculosis care. Wkly Epidemiol Rec 2006;81:43-7.

45. Achkar JM, Sherpa T, Cohen HW, Holzman RS. Differences

in clinical presentation among persons with pulmonary

tuberculosis: a comparison of documented and undocu-

mented foreign-born versus US-born persons. Clin Infect Dis

2008;47:1277-83.

46. Dewan PK, Grinsdale J, Kawamura LM. Low sensitivity of a whole-blood interferon-gamma release assay for detection of active tuberculosis. Clin Infect Dis 2007;44:69-73.

47. Hopewell PC, Pai M, Maher D, Uplekar M, Raviglione MC. In- ternational standards for tuberculosis care. Lancet Infect Dis

2006;6:710-25.

48. Tattevin P, Che D, Fraisse P, Gatey C, Guichard C, Antoine D,

et al. Factors associated with patient and health care system

delay in the diagnosis of tuberculosis in France. Int J Tuberc

Lung Dis 2012;16:510-5.